New techniques for determining base sequences of DNA or RNA have been implemented.
In a method using the conventional electrophoresis, a sample of DNA fragments for sequencing or cDNA fragments synthesized by reverse transcription from an RNA sample is prepared in advance and a dideoxy reaction is carried out by performing the known Sanger sequencing technology. Thereafter, electrophoresis is performed and a molecular weight separation and development pattern is measured for analysis.
Contrary to this, recently, a method has been proposed whereby sequence information of a number of fragments is determined in parallel by fixing a number of sample DNA fragments to a substrate.
In NPL 1, a microparticle is used as a carrier for carrying DNA fragments and PCR is performed on the microparticle. Thereafter, the microparticles supporting the DNA fragments, having been amplified by PCR, are added to a plate provided with a number of holes having a hole diameter matched with the size of the microparticle, thereby reading the DNA fragments by the pyrosequencing method.
Also in NPL 2, a microparticle is used as a carrier for carrying DNA fragments and PCR is performed on the microparticle. Thereafter, the microparticles are spread over a glass substrate and fixed thereto. An enzyme reaction (ligation) is carried out on the glass substrate and a primer attached with a florescent dye is allowed to be incorporated, whereby performing florescence detection allows for obtaining sequence information on each of the fragments.
Furthermore in NPL 3, a number of DNA probes having the same sequence are fixed to a substrate. Also, after cleaving a DNA sample, an adapter sequence of a complementary chain to a sequence of the DNA probe is added to a terminal of each of the sample DNA fragments. Subjecting the above to hybridization on the substrate allows for fixing the sample DNA fragments molecule by molecule to the substrate in a random manner. In this case, a DNA elongation reaction is carried out on the substrate and a substrate attached with a florescent dye is allowed to be incorporated. Thereafter, washing off unreacted substrates or florescence detection is performed and sequence information of the sample DNA is obtained.
In this manner, a method has been developed and implemented whereby sequence information of a number of fragments is determined in parallel by fixing a number of sample nucleic acid fragments to a substrate.
PTL 1 discloses details of a method for determining a number of pieces of sequence information in parallel. A sequence reaction is carried out in a flow cell having a flow passage therein. A surface of the flow passage is coated with acrylamide and a forward primer and a reverse primer are grafted on the acrylamide. A solution containing target templates of a plurality of types is introduced into the flow cell and then an amplification reaction is carried out on a substrate by subjecting the flow cell to temperature cycling, thereby forming, on the substrate, a plurality of colonies having the same sequence as the template. One colony is a collection of a plurality of copy templates having been amplified while the copy template has the same sequence as or a complementary sequence to the target template. After amplification, a sequencing primer is hybridized with the template in the colony, thereby allowing the sequence reaction to be carried out. For the sequence reaction, a nucleotide disclosed in WO 2004/018493 is used. The nucleotide has a florescent molecule via a cleavable linker and a leaving group at 3′-oxygen of its sugar. In the sequence reaction, four types of nucleotides derived from four types of bases A, T, C, and G are used. Each of the four types of nucleotides is labeled with a florescent dye different from each another. A solution containing a polymerase and the four types of nucleotides is injected into the flow cell to allow the sequence primer to incorporate the nucleotides. Thereafter, the sequence primer incorporated with the florescence is subjected to florescence detection. Here, since the nucleotides are incorporated in such a manner as to form a complementary sequence to the template sequence, detecting florescence of the nucleotides having been incorporated allows for determining a base sequence of the target template. After the florescence detection, the intramolecular linker is chemically cleaved to remove the florescent molecule. By repeating a cycle of incorporation of the nucleotides, florescence detection, removal of the florescent molecule, the base sequence of the template is determined.